Hybrid maize plant and seed 38D66

ABSTRACT

According to the invention, there is provided a hybrid maize plant, designated as 38D66, produced by crossing two Pioneer Hi-Bred International, Inc. proprietary inbred maize lines. This invention relates to the hybrid seed 38D66, the hybrid plant produced from the seed, and variants, mutants, and trivial modifications of hybrid 38D66.

FIELD OF THE INVENTION

This invention is in the field of maize breeding, specifically relatingto hybrid maize designated 38D66.

BACKGROUND OF THE INVENTION Plant Breeding

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Maize (Zea mays L.), often referred to as corn in the United States, canbe bred by both self-pollination and cross-pollination techniques. Maizehas separate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears.

The development of a hybrid maize variety involves three steps: (1) theselection of plants from various germplasm pools for initial breedingcrosses; (2) the selfing of the selected plants from the breedingcrosses for several generations to produce a series of inbred lines,which, although different from each other, breed true and are highlyuniform; and (3) crossing the selected inbred lines with unrelatedinbred lines to produce the hybrid progeny (F1). During the inbreedingprocess in maize, the vigor of the lines decreases. Vigor is restoredwhen two different inbred lines are crossed to produce the hybridprogeny (F1). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid created by crossing adefined pair of inbreds will always be the same. Once the inbreds thatcreate a superior hybrid have been identified, a continual supply of thehybrid seed can be produced using these inbred parents and the hybridcorn plants can then be generated from this hybrid seed supply.

Large scale commercial maize hybrid production, as it is practicedtoday, requires the use of some form of male sterility system whichcontrols or inactivates male fertility. A reliable method of controllingmale fertility in plants also offers the opportunity for improved plantbreeding. This is especially true for development of maize hybrids,which relies upon some sort of male sterility system. There are severaloptions for controlling male fertility available to breeders, such as:manual or mechanical emasculation (or detasseling), cytoplasmic malesterility, genetic male sterility, gametocides and the like.

Hybrid maize seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twoinbred varieties of maize are planted in a field, and the pollen-bearingtassels are removed from one of the inbreds (female) prior to pollenshed. Providing that there is sufficient isolation from sources offoreign maize pollen, the ears of the detasseled inbred will befertilized only from the other inbred (male), and the resulting seed istherefore hybrid and will form hybrid plants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent inmaize plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Usually seedfrom detasseled fertile maize and CMS produced seed of the same hybridare blended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., of Pioneer Hi-Bred, U.S. patentapplication Ser. No. 07/848,433, have developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility; silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not "on"resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning "on", the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran antisense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see:Fabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, Glenn R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach.

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. The development of maize hybrids requires, in general,the development of homozygous inbred lines, the crossing of these lines,and the evaluation of the crosses. Breeding programs combine the geneticbackgrounds from two or more inbred lines or various other broad-basedsources into breeding pools from which new inbred lines are developed byselfing and selection of desired phenotypes. Hybrids also are used asstarting materials for breeding or source populations from which todevelop inbred lines. The inbred lines derived from hybrids can bedeveloped using methods of breeding known in the art such as pedigreebreeding and recurrent selection. New inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which of those have commercial potential.

Recurrent selection breeding, backcrossing for example, can be used toimprove inbred lines and a hybrid which is made using those inbreds.Backcrossing can be used to transfer a specific desirable trait from oneinbred or source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred(recurrent parent) to a donor inbred (non-recurrent parent), thatcarries the appropriate gene(s) for the trait in question. The progenyof this cross is then mated back to the superior recurrent parentfollowed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait and for thegermplasm inherited from the recurrent parent, the progeny will behomozygous for loci controlling the characteristic being transferred,but will be like the superior parent for essentially all other genes.The last backcross generation is then selfed to give pure breedingprogeny for the gene(s) being transferred. A hybrid developed frominbreds containing the transferred gene(s) is essentially the same as ahybrid developed from the same inbreds without the transferred gene(s).

There are many important factors to be considered in the art of plantbreeding, such as the ability to recognize important morphological andphysiological characteristics, the ability to design evaluationtechniques for genotypic and phenotypic traits of interest, and theability to search out and exploit the genes for the desired traits innew or improved combinations.

The objective of commercial maize hybrid line development programs is todevelop new inbred lines to produce hybrids that combine to produce highgrain yields and superior agronomic performance. The primary traitbreeders seek is yield. However, many other major agronomic traits areof importance in hybrid combination and have an impact on yield orotherwise provide superior performance in hybrid combinations. Suchtraits include percent grain moisture at harvest, relative maturity,resistance to stalk breakage, resistance to root lodging, grain quality,and disease and insect resistance. In addition, the lines per se musthave acceptable performance for parental traits such as seed yields,kernel sizes, pollen production, all of which affect ability to provideparental lines in sufficient quantity and quality for hybridization.These traits have been shown to be under genetic control and many if notall of the traits are affected by multiple genes.

Pedigree Breeding

The pedigree method of breeding is the mostly widely used methodologyfor new hybrid line development.

In general terms this procedure consists of crossing two inbred lines toproduce the non-segregating F1 generation, and self pollination of theF1 generation to produce the F2 generation that segregates for allfactors for which the inbred parents differ. An example of this processis set forth below. Variations of this generalized pedigree method areused, but all these variations produce a segregating generation whichcontains a range of variation for the traits of interest.

EXAMPLE 1 Hypothetical Example of Pedigree Breeding Program

Consider a cross between two inbred lines that differ for alleles at sixloci.

The parental genotypes are:

    ______________________________________    Parent 1         A b C d e F/A b C d e F    Parent 2         a B c D E f/a B c D E f    ______________________________________

the F1 from a cross between these two parents is:

    ______________________________________    F1              A b C d e F/a B c D E f    ______________________________________

Selfing F1 will produce an F2 generation including the followinggenotypes:

    ______________________________________    A B c D E f/a b C d e F    A B c D e f/a b C d E F    A B c D e f/a b C d e F    ______________________________________

The number of genotypes in the F2 is 36 for six segregating loci (729)and will produce (26)-2 possible new inbreds, (62 for six segregatingloci).

Each inbred parent which is used in breeding crosses represents a uniquecombination of genes, and the combined effects of the genes define theperformance of the inbred and its performance in hybrid combination.There is published evidence (Smith, O. S., J. S. C. Smith, S. L. Bowen,R. A. Tenborg and S. J. Wall, TAG 80: 833-840 (1990)) that each of thelines are different and can be uniquely identified on the basis ofgenetically-controlled molecular markers.

It has been shown (Hallauer, Arnel R. and Miranda, J. B. Fo.Quantitative Genetics in Maize Breeding, Iowa State University Press,Ames Iowa, 1981) that most traits of economic value in maize are underthe genetic control of multiple genetic loci, and that there are a largenumber of unique combinations of these genes present in elite maizegermplasm. If not, genetic progress using elite inbred lines would nolonger be possible. Studies by Duvick and Russell (Duvick, D. N.,Maydica 37: 69-79, (1992); Russell, W. A., Maydica XXIX: 375-390 (1983))have shown that over the last 50 years the rate of genetic progress incommercial hybrids has been between one and two percent per year.

The number of genes affecting the trait of primary economic importancein maize, grain yield, has been estimated to be in the range of 10-1000.Inbred lines which are used as parents for breeding crosses differ inthe number and combination of these genes. These factors make the plantbreeder's task more difficult. Compounding this is evidence that no oneline contains the favorable allele at all loci, and that differentalleles have different economic values depending on the geneticbackground and field environment in which the hybrid is grown. Fiftyyears of breeding experience suggests that there are many genesaffecting grain yield and each of these has a relatively small effect onthis trait. The effects are small compared to breeders'ability tomeasure grain yield differences in evaluation trials. Therefore, theparents of the breeding cross must differ at several of these loci sothat the genetic differences in the progeny will be large enough thatbreeders can develop a line that increases the economic worth of itshybrids over that of hybrids made with either parent.

If the number of loci segregating in a cross between two inbred lines isn, the number of unique genotypes in the F2 generation is 3n and thenumber of unique inbred lines from this cross is {(2n)-2}. Only a verylimited number of these combinations are useful. Only about 1 in 10,000of the progeny from F2's are commercially useful.

By way of example, if it is assumed that the number of segregating lociin F2 is somewhere between 20 and 50, and that each parent is fixed forhalf the favorable alleles, it is then possible to calculate theapproximate probabilities of finding an inbred that has the favorableallele at {(n/2)+m} loci, where n/2 is the number of favorable allelesin each of the parents and m is the number of additional favorablealleles in the new inbred. See Example 2 below. The number m is assumedto be greater than three because each allele has so small an effect thatevaluation techniques are not sensitive enough to detect differences dueto three or less favorable alleles. The probabilities in Example 2 areon the order of 10-5 or smaller and they are the probabilities that atleast one genotype with (n/2)=m favorable alleles will exist.

To put this in perspective, the number of plants grown on 60 millionacres (approximate United States corn acreage) at 25,000 plants/acre is1.5×1012.

EXAMPLE 2 Probability of Finding an Inbred with m of n FavorableAlleles.

Assume each parent has n/2 of the favorable alleles and only 1/2 of thecombinations of loci are economically useful.

    ______________________________________    No. of   No. of favorable                         No. additional    segregating             alleles in Parents                         favorable alleles                                     Probability that    loci (n) (n/2)       in new inbred                                     genotype occurs*    ______________________________________    20       10          14          3 × 10-5    24       12          16          2 × 10-5    28       14          18          1 × 10-5    32       16          20          8 × 10-6    36       18          22          5 × 10-6    40       20          24          3 × 10-6    44       22          26          2 × 10-6    48       24          28          1 × 10-6    ______________________________________     *Probability that a useful combination exists, does not include the     probability of identifying this combination if it does exist.

The possibility of having a usably high probability of being able toidentify this genotype based on replicated field testing would be mostlikely smaller than this, and is a function of how large a population ofgenotypes is tested and how testing resources are allocated in thetesting program.

SUMMARY OF THE INVENTION

According to the invention, there is provided a hybrid maize plant,designated as 38D66, produced by crossing two Pioneer Hi-BredInternational, Inc. proprietary inbred maize lines. This invention thusrelates to the hybrid seed 38D66, the hybrid plant produced from theseed, and variants, mutants and trivial modifications of hybrid 38D66.This invention also relates to methods for producing a maize plantcontaining in its genetic material one or more transgenes and to thetransgenic maize plants produced by that method. This invention furtherrelates to methods for producing maize lines derived from hybrid maizeline 38D66 and to the maize lines derived by the use of those methods.This hybrid maize is characterized by consistent high yield for itsmaturity and exceptional late season plant health. 38D66 further hasvery fast dry down and dependable drought tolerance.

Definitions

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. NOTE: ABS is in absolute termsand %MN is percent of the mean for the experiments in which the inbredor hybrid was grown. These designators will follow the descriptors todenote how the values are to be interpreted. Below are the descriptorsused in the data tables included herein.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance.

BAR PLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BRT STK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap in pairedcomparisons and on a 1 to 9 scale (9=highest resistance) inCharacteristics Charts.

BU ACR=YIELD (BUSHELS/ACRE). Yield of the grain at harvest in bushelsper acre adjusted to 15.5% moisture.

CLN=CORN LETHAL NECROSIS (synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV)). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance.

COM RST=COMMON RUST (Puccinia sorghi). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance.

CRM=COMPARATIVE RELATIVE MATURITY (see PRM).

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1-9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

D/E=DROPPED EARS. Represented in a 1 to 9 scale in the CharacteristicsChart, where 9 is the rating representing the least, or no, droppedears.

DIP ERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance.

DRP EAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest.

D/T=DROUGHT TOLERANCE. This represents a 1-9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance.

EAR HT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured in inches.This is represented in a 1 to 9 scale in the Characteristics Chart,where 9 is highest.

EAR MLD=General Ear Mold. Visual rating (1-9 score) where a "1" is verysusceptible and a "9" is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold.

EAR SZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

ECB 1LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance.

ECB 2IT=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilalis). Average inches of tunneling per plant in thestalk.

ECB 2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by European Corn Borer, Second Generation.A higher score indicates a higher resistance.

ECB DPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation corn borer infestation.

E/G=EARLY GROWTH. This represents a 1 to 9 rating for early growth,scored when two leaf collars are visible.

EST CNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYE SPT=Eye Spot (Kabatiella zeae or Aureobasidium zeae). A 1 to 9visual rating indicating the resistance to Eye Spot. A higher scoreindicates a higher resistance.

FUS ERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium ear rot. A higher score indicates a higher resistance.

G/A=GRAIN APPEARANCE. Appearance of grain in the grain tank (scored downfor mold, cracks, red streak, etc.).

GDU=Growing Degree Units. Using the Barger Heat Unit Theory, thatassumes that maize growth occurs in the temperature range 50° F.-86° F.and that temperatures outside this range slow down growth; the maximumdaily heat unit accumulation is 36 and the minimum daily heat unitaccumulation is 0. The seasonal accumulation of GDU is a major factor indetermining maturity zones.

GDU PHY=GDU TO PHYSIOLOGICAL MATURITY. The number of growing degreeunits required for an inbred or hybrid line to have approximately 50percent of plants at physiological maturity from time of planting.Growing degree units are calculated by the Barger method.

GDU SHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24-hour period are: ##EQU1##

The highest maximum temperature used is 86° F. and the lowest minimumtemperature used is 50° F. For each inbred or hybrid it takes a certainnumber of GDUs to reach various stages of plant development.

GDU SLK=GDU TO SILK. The number of growing degree units required for aninbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GIB ERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae). A 1 to 9visual rating indicating the resistance to Gibberella Ear Rot. A higherscore indicates a higher resistance.

GLF SPT=Gray Leaf Spot (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance. GOS WLT=Goss' Wilt (Corynebacterium nebraskense). A 1to 9 visual rating indicating the resistance to Goss' Wilt. A higherscore indicates a higher resistance.

GRN APP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain quality.

H/POP=YIELD AT HIGH DENSITY. Yield ability at relatively high plantdensities on 1-9 relative rating system with a higher number indicatingthe hybrid responds well to high plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to increased plant density.

HC BLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporiumcarbonum). A 1 to 9 visual rating indicating the resistance toHelminthosporium infection. A higher score indicates a higherresistance.

HD SMT=Head Smut (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridon per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. GROSS INCOME advantage of variety #1over variety #2.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1-9 relative system with a higher number indicating thehybrid responds well to low plant densities for yield relative to otherhybrids. A 1, 5, and 9 would represent very poor, average, and very goodyield response, respectively, to low plant density.

MDM CPX=Maize Dwarf Mosaic Complex (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance.

MST=HARVEST MOISTURE. The moisture is the actual percentage moisture ofthe grain at harvest.

MST ADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2--MOISTURE of variety#1=MOISTURE ADVANTAGE of variety #1.

NLF BLT=Northern Leaf Blight (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance.

OIL=GRAIN OIL. The amount of the kernel that is oil, expressed as apercentage on a dry weight basis.

PHY CRM=CRM at physiological maturity.

PLT HT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in inches. This is represented as a1 to 9 scale, 9 highest, in the Characteristics Chart.

POL SC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POL WT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

POP K/A=PLANT POPULATIONS. Measured as 1000s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2--PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED Relative Maturity. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRM SHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PRO=PROTEIN RATING . Rating on a 1 to 9 scale comparing relative amountof protein in the grain compared to hybrids of similar maturity. A "1"score difference represents a 0.4 point change in grain protein percent(e.g., 8.0% to 8.4%).

PROTEIN=GRAIN PROTEIN. The amount of the kernel that is crude protein,expressed as a percentage on a dry weight basis.

P/Y=PROTEIN/YIELD RATING. Indicates, on a 1 to 9 scale, the economicvalue of a hybrid for swine and poultry feeders. This takes into accountthe income due to yield, moisture and protein content.

ROOTS (%)=Percent of stalks NOT root lodged at harvest.

R/L=ROOT LODGING. A 1 to 9 rating indicating the level of root lodgingresistance. The higher score represents higher levels of resistance.

RT LDG=ROOT LODGING. Root lodging is the percentage of plants that donot root lodge; plants that lean from the vertical axis as anapproximately 30° angle or greater would be counted as root lodged.

RTL ADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2.

S/L=STALK LODGING. A 1 to 9 rating indicating the level of stalk lodgingresistance. The higher scores represent higher levels of resistance.

SCT GRN=SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter grain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SDG VGR=SEEDLING VIGOR. This is the visual rating (1 to 9) of the amountof vegetative growth after emergence at the seedling stage(approximately five leaves). A higher score indicates better vigor.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SLF BLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance.

SLK CRM=CRM at Silking.

SOU RST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance.

STA GRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STAND (%)=Percent of stalks standing at harvest.

STARCH=GRAIN STARCH. The amount of the kernel that is starch, expressedas a percentage on a dry weight basis.

STD ADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STK CNT=NUMBER OF PLANTS. This is the final stand or number of plantsper plot.

STK LDG=STALK LODGING. This is the percentage of plants that did notstalk lodge (stalk breakage) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear.

STW WLT=Stewart's Wilt (Erwinia stewartii). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance.

TAS BLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting.

TAS SZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TEX EAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TIL LER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TST WT (CHARACTERISTICS CHART)=Test weight on a 1 to 9 rating scale witha 9 being the highest rating.

TST WT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grainin pounds for a given volume (bushel).

TST WTA=TEST WEIGHT ADJUSTED. The measure of the weight of the grain inpounds for a given volume (bushel) adjusted for 15.5 percent moisture.

TSW ADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M%=PERCENT MOISTURE WINS.

WIN Y%=PERCENT YIELD WINS.

YLD=YIELD. It is the same as BU ACR ABS.

YLD ADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1--YIELD variety #2=yieldadvantage of variety #1.

YLD SC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

DETAILED DESCRIPTION OF THE INVENTION

Pioneer Brand Hybrid 38D66 has consistent high yield. The hybrid showsstrong and very fast dry-down and consistent drought tolerance. 38D66exhibits consistent brittle stalk breakage tolerance and exceptionallate season plant health. It is particularly suited to the Northcentral,Northeast, Northwestern and Western United States as well as Canada. Thevariety has broad adaptability.

Pioneer Brand Hybrid 38D66 is a single cross, yellow endosperm, dentmaize hybrid. Hybrid 38D66 has a relative maturity of approximately 91based on the Comparative Relative Maturity Rating System for harvestmoisture of grain.

This hybrid has the following characteristics based on the datacollected primarily at Johnston, Iowa.

                  TABLE 1    ______________________________________    VARIETY DESCRIPTION INFORMATION    VARIETY = 38D66    ______________________________________    1. TYPE: (describe intermediate types in Comments section):    2     1 = Sweet 2 = Dent 3 = Flint 4 = Flour 5 = Pop 6 = Ornamental    2. MATURITY:          HEAT    DAYS  UNITS    064           1,098.0 From emergence to 50% of plants in silk    064           1,098.0 From emergence to 50% of plants in pollen    004           0,094.5 From 10% to 90% pollen shed    065           1,450.0 From 50% silk to harvest at 25% moisture    3. PLANT:               Standard Sample                            Deviation                                     Size    0,272.5          cm      Plant Height (to tassel tip)                                    3.54    2    0,142.5          cm      Ear Height (to base of top ear                                    3.54    2                  node)    0,017.5          cm      Length of Top Ear Internode                                    3.82   10    0.0           Average Number of Tillers                                    0.00    2    1.0           Average Number of Ears per                                    0.00    2                  Stalk    1.0           Anthocyanin of Brace Roots:            1 = Absent 2 = Faint 3 = Moderate 4 = Dark    4. LEAF:                Standard Sample                            Deviation                                     Size    009.3 cm      Width of Ear Node Leaf                                    1.27   10    087.5 cm      Length of Ear Node Leaf                                    3.54   10    05.8          Number of leaves above top ear                                    0.85   10    029.5         Degrees Leaf Angle (measure                                    0.71    2                  from 2nd leaf above ear                  at anthesis to stalk above leaf)    03            Leaf Color Dark Green (Munsell code) 5GY34    1.0           Leaf Sheath Pubescence (Rate on scale from                  1 = none to 9 = like peach fuzz)    7.5           Marginal Waves (Rate on scale from                  1 = none to 9 = many)    3.0           Longitudinal Creases (Rate on scale from                  1 = none to 9 = many)    5. TASSEL:              Standard Sample                            Deviation                                     Size    07.9          Number of Primary Lateral                                    0.14   10                  Branches    032.0         Branch Angle from Central                                    1.41    2                  Spike    62.0  cm      Tassel Length     0.85    2                  (from top leaf collar to tassel tip)    7.0           Pollen Shed            (rate on scale from 0 = male sterile to 9 = heavy shed)    11            Anther Color Pink (Munsell code) 7.5R64    01            Glume Color Light Green (Munsell code) 2.5GY66    1.0           Bar Glumes (Glume Bands): 1 = Absent 2 = Present    25            Peduncle Length (cm. from top leaf to basal branches)    6a. EAR (Unhusked Data):    1   Silk Color (3 days after emergence) Light Green        (Munsell code) 2.5GY88    2   Fresh Husk Color (25 days after 50% silking)        Medium Green (Munsell code) 5GY56    21  Dry Husk Color (65 days after 50% silking) Buff        (Munsell code) 2.5Y84    1   Position of Ear at Dry Husk Stage:        1 = Upright 2 = Horizontal 3 = Pendant Upright    5   Husk Tightness (Rate of Scale from 1 = very loose to 9 = very tight)    1   Husk Extension (at harvest): 1 = Short (ears exposed)        2 = Medium (<8 cm) 3 = Long (8-10 cm beyond ear tip)        4 = Very Long (>10 cm) Short    6b. EAR (Husked Ear Data):                            Standard Sample                            Deviation                                     Size    18    cm      Ear Length        0.00   10    46    mm      Ear Diameter at mid-point                                    0.71   10    189   gm      Ear Weight        4.95   10    15            Number of Kernel Rows                                    0.00   10    2             Kernel Rows: 1 = Indistinct 2 = Distinct Distinct    2             Row Alignment: 1 = Straight                  2 = Slightly Curved 3 = Spiral Slightly Curved    12    cm      Shank Length      1.41   10    2             Ear Taper: 1 = Slight            2 = Average 3 = Extreme Average    7. KERNEL (Dried):      Standard Sample                            Deviation                                     Size    14    mm      Kernel Length     0.00   10    8     mm      Kernel Width      0.00   10    4     mm      Kernel Thickness  0.00   10    11            % Round Kernels (Shape Grade)                                    3.54    2    1             Aleurone Color Pattern:            1 = Homozygous 2 = Segregating Homozygous    7             Aluerone Color Yellow (Munsell code) 10YR712    7             Hard Endosperm Color Yellow (Munsell code)                  10YR712    3             Endosperm Type: Normal Starch                  1 = Sweet (Su1) 2 = Extra Sweet (sh2)                  3 = Normal Starch 4 = High Amylose Starch                  5 = Waxy Starch 6 = High Protein                  7 = High Lysine 8 = Super Sweet (se) 9 = High Oil                  10 = Other --    33    gm      Weight per 100 Kernels                                    0.71    2                  (unsized sample)    8. COB:                 Standard Sample                            Deviation                                     Size    23    mm      Cob Diameter at mid-point                                    0.71   10    23            Cob Color Brown (Munsell code) 10R36    9. DISEASE RESISTANCE    (Rate from 1 (most susceptible) to 9 (most resistant); leave blank    if not tested; leave Race or Strain Options blank if polygenic):    A. Leaf Blights, Wilts, and Local Infection Diseases          Anthracnose Leaf Blight (Colletotrichum graminicola)          Common Rust (Puccinia sorghi)          Common Smut (Ustilago maydis)    7     Eyespot (Kabatiella zeae)    8     Goss's Wilt (Clavibacter michiganense spp. nebraskense)          Gray Leaf Spot (Cercospora zeae-maydis)          Helminthosporium Leaf Spot (Bipolaris zeicola) Race --    7     Northern Leaf Blight (Exserohilum turcicum) Race --    7     Southern Leaf Blight (Bipolaris maydis) Race --          Southern Rust (Puccinia polysora)          Stewart's Wilt (Erwinia stewartii)          Other (Specify) --    B. Systemic Diseases    Corn Lethal Necrosis (MCMV and MDMV)    Head Smut (Sphacelotheca reiliana)    Maize Chlorotic Dwarf Virus (MDV)    Maize Chlorotic Mottle Virus (MCMV)    Maize Dwarf Mosaic Virus (MDMV)    Sorghum Downy Mildew of Corn (Peronosclerospora sorghi)    Other (Specify) --    C. Stalk Rots          Anthracnose Stalk Rot (Colletotrichum graminicola)          Diplodia Stalk Rot (Stenocarpella maydis)          Fusarium Stalk Rot (Fusarium moniliforme)          Gibberella Stalk Rot (Gibberella zeae)          Other (Specify) --    D. Ear and Kernel Rots          Aspergillus Ear and Kernel Rot (Aspergillus flavus)          Diplodia Ear Rot (Stenocarpella maydis)          Fusarium Ear and Kernel Rot (Fusarium moniliforme)    5     Gibberella Ear Rot (Gibberella zeae)          Other (Specify) --          Banks grass Mite (Oligonychus pratensis)          Corn Worm (Helicoverpa zea)          Leaf Feeding          Silk Feeding          mg larval wt.          Ear Damage          Corn Leaf Aphid (Rhopalosiphum maidis)          Corn Sap Beetle (Carpophilus dimidiatus)          European Corn Borer (Ostrinia nubilalis)    4     1st Generation (Typically Whorl Leaf Feeding)          2nd Generation (Typically Leaf Sheath-Collar Feeding)          Stalk Tunneling          cm tunneled/plant          Fall Armyworm (Spodoptera fruqiperda)          Leaf Feeding          Silk Feeding          mg larval wt.          Maize Weevil (Sitophilus zeamaize)          Northern Rootworm (Diabrotica barberi)          Southern Rootworm (Diabrotica undecimpunctata)          Southwestern Corn Borer (Diatreaea grandiosella)          Leaf Feeding          Stalk Tunneling          cm tunneled/plant          Two-spotted Spider Mite (Tetranychus urticae)          Western Rootworm (Diabrotica virgifrea virgifera)          Other (Specify) --    11. AGRONOMIC TRAITS:    6     Staygreen (at 65 days after anthesis)          (Rate on a scale from 1 = worst to 9 = excellent)    0.3   % Dropped Ears (at 65 days after anthesis)          % Pre-anthesis Brittle Snapping          % Pre-anthesis Root Lodging    8.3   Post-anthesis Root Lodging (at 65 days after anthesis)    9,474 Kg/ha Yield (at 12-13% grain moisture)    ______________________________________     *In interpreting the foregoing color designations, reference may be had t     the Munsell Glossy Book of Color, a standard color reference.

Research Comparisons for Pioneer Hybrid 38D66

Comparisons of characteristics for Pioneer Brand Hybrid 38D66 were madeagainst Pioneer Brand Hybrids 3861, 3893, 38R21, and 3860.

Table 2A compares Pioneer Hybrid 38D66 and Pioneer Brand Hybrid 3861, ahybrid of similar maturity. The results show that 38D66 hassignificantly lower harvest moisture than hybrid 3861. Hybrid 38D66 alsodemonstrates significantly superior stay green and significantly shorterplant height with significantly higher ear placement than hybrid 3861.38D66 also is earlier to flower with significantly fewer growing degreeunits to silk and a significantly lower predicted relative maturityscore than hybrid 3861.

Table 2B compares Pioneer Hybrid 38D66 and Pioneer Hybrid 3893, a hybridof similar maturity. The results indicate that hybrid 38D66 issignificantly higher yielding than the 3893 hybrid. The 38D66 hybridalso demonstrates significantly taller plant stature than the 3893hybrid. Hybrid 38D66 further is later to flower with significantlyhigher number of growing degree units to pollen shed and to silk and asignificantly higher predicted relative maturity score than hybrid 3893.Hybrid 38D66 exhibits higher brittle snap breakage resistance than 3893.Hybrid 38D66 also exhibits superior tolerance to head smut than 3893.

Table 2C compares Pioneer Hybrid 38D66 and Pioneer Hybrid 38R21, ahybrid of similar maturity. The results show that Hybrid 38D66demonstrates significantly superior resistance to root lodging thanhybrid 38R21. The 38D66 hybrid demonstrates significantly shorter plantstature with significantly lower ear placement than hybrid 38R21. 38D66also is later to flower with a significantly higher number of growingdegree units to pollen shed and to silk than hybrid 38R21.

Table 2D compares Pioneer Hybrid 38D66 and Pioneer Hybrid 3860, a hybridof similar maturity. The results show that Hybrid 38D66 demonstrates asignificantly higher early stand count as well as significantly tallerplant stature than hybrid 38D66. Hybrid 38D66 also demonstratessignificantly superior resistance to brittle snap and a significantlylarger stalk count than hybrid 3860. The 38D66 hybrid is also later toflower with a significantly larger number of growing degree units topollen shed than hybrid 3860.

                                      TABLE 2A    __________________________________________________________________________    HYBRID COMPARISON REPORT    VARIETY #1 = 38D66    VARIETY #2 = 3861    __________________________________________________________________________                   PRM BU  BU      TST SDG EST GDU               PRM SHD ACR ACR MST WT  VGR CNT SHD               ABS ABS ABS % MN                               % MN                                   ABS % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   91  95  152.1                           103 99  52.6                                       98  102 102           2   93  94  150.6                           102 103 52.5                                       100 102 102           LOCS               15  18  114 114 114 50  71  70  54           REPS               15  18  163 163 158 72  96  86  80           DIFF               2   1   1.4 1   4   0.1 3   0   0           PR > T               .000#                   .171                       .403                           .331                               .000#                                   .535                                       .377                                           .999                                               .999    __________________________________________________________________________               GDU STK PLT EAR RT  STA STK BRT DRP               SLK CNT HT  HT  LDG GRN LDG STK EAR               % MN                   % MN                       % MN                           % MN                               % MN                                   % MN                                       % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   100 101 101 103 99  102 98  103 100           2   102 101 104 99  102 84  100 102 100           LOCS               21  142 49  49  47  54  93  31  31           REPS               31  211 74  74  63  70  129 40  41           DIFF               2   0   3   5   3   18  1   1   0           PR > T               .000#                   .999                       .001#                           .010+                               .168                                   .003#                                       .305                                           .611                                               .999    __________________________________________________________________________               NLF GOS HD  GIB EYE ECB ECB               BLT WLT SMT ERS SPT DPE 1LF               ABS ABS ABS ABS ABS ABS ABS    __________________________________________________________________________    TOTAL SUM           1   7.5 8.3 100.0                           4.7 6.5 94.3                                       4.0           2   4.0 7.5 98.6                           4.7 7.3 90.4                                       6.0           LOCS               1   2   4   3   2   7   1           REPS               2   3   10  6   4   13  1           DIFF               3.5 0.8 1.4 0.0 0.8 3.8 2.0           PR > T  .500                       .182                           .999                               .205                                   .093*    __________________________________________________________________________     * = 10% SIG     + = 5% SIG     # = 1% SIG

                                      TABLE 2B    __________________________________________________________________________    HYBRID COMPARISON REPORT    VARIETY #1 = 38D66    VARIETY #2 = 3893    __________________________________________________________________________                   PRM BU  BU      TST SDG EST GDU               PRM SHD ACR ACR MST WT  VGR CNT SHD               ABS ABS ABS % MN                               % MN                                   ABS % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   91  95  153.3                           103 99  52.4                                       97  102 102           2   89  90  149.7                           100 96  53.4                                       97  102 98           LOCS               15  22  114 114 114 47  74  65  61           REPS               15  22  168 168 163 69  107 81  95           DIFF               2   5   3.6 3   4   1.0 0   1   4           PR > T               .005#                   .000#                       .022+                           .020+                               .000#                                   .000#                                       .999                                           .598                                               .000#    __________________________________________________________________________               GDU STK PLT EAR RT  STA STK BRT DRP               SLK CNT HT  HT  LDG GRN LDG STK EAR               % MN                   % MN                       % MN                           % MN                               % MN                                   % MN                                       % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   101 101 102 104 98  102 98  103 100           2   99  100 98  103 100 101 99  98  100           LOCS               27  149 57  57  48  56  91  32  26           REPS               44  231 91  91  66  77  131 41  36           DIFF               2   0   3   1   2   1   1   5   0           PR > T               .000#                   .999                       .000#                           .552                               .573                                   .934                                       .352                                           .082*                                               .999    __________________________________________________________________________               NLF SLF GOS HD  GIB EYE ECB ECB               BLT BLT WLT SMT ERS SPT DPE 1LF               ABS ABS ABS ABS ABS ABS ABS ABS    __________________________________________________________________________    TOTAL SUM           1   6.6 6.5 8.3 100.0                               4.7 6.5 94.3                                           4.0           2   5.4 7.5 8.3 96.0                               4.8 6.0 89.8                                           4.0           LOCS               5   1   2   4   3   2   7   1           REPS               10  2   3   10  6   4   13  1           DIFF               1.2 1.0 0.0 4.0 0.2 0.5 4.4 0.0           PR > T               .178    .999                           .038+                               .808                                   .500                                       .090*    __________________________________________________________________________     * = 10% SIG     + = 5% SIG     # = 1% SIG

                                      TABLE 2C    __________________________________________________________________________    HYBRID COMPARISON REPORT    VARIETY #1 = 38D66    VARIETY #2 = 38R21    __________________________________________________________________________                   PRM BU  BU      TST SDG EST GDU               PRM SHD ACR ACR MST WT  VGR CNT SHD               ABS ABS ABS % MN                               % MN                                   ABS % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   91  95  153.2                           100 101 52.1                                       94  102 102           2   90  92  161.1                           105 97  52.4                                       106 101 99           LOCS               9   15  84  84  84  38  51  45  48           REPS               9   15  125 125 121 56  72  56  71           DIFF               1   3   7.9 5   3   0.3 11  1   2           PR > T               .312                   .000#                       .000#                           .000#                               .000#                                   .313                                       .002#                                           .540                                               .000#    __________________________________________________________________________               GDU STK PLT EAR RT  STA STK BRT DRP               SLK CNT HT  HT  LDG GRN LDG STK EAR               % MN                   % MN                       % MN                           % MN                               % MN                                   % MN                                       % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   100 101 101 103 100 99  98  102 100           2   98  100 103 111 93  92  101 100 100           LOCS               19  112 43  43  32  49  72  18  22           REPS               27  178 69  69  44  68  104 25  31           DIFF               2   1   2   8   7   7   3   1   0           PR > T               .001#                   .156                       .000#                           .000#                               .011+                                   .313                                       .014+                                           .255                                               .999    __________________________________________________________________________               NLF GOS HD  GIB EYE ECB ECB               BLT WLT SMT ERS SPT DPE 1LF               ABS ABS ABS ABS ABS ABS ABS    __________________________________________________________________________    TOTAL SUM           1   7.5 8.3 100.0                           4.7 6.5 98.3                                       4.0           2   5.0 7.0 100.0                           5.8 5.8 98.5                                       4.0           LOCS               1   2   4   3   2   4   1           REPS               2   3   10  6   4   8   1           DIFF               2.5 1.3 0.0 1.2 0.8 0.2 0.0           PR > T  .126                       .999                           .020+                               .205                                   .791    __________________________________________________________________________     * = 10% SIG     + = 5% SIG     # = 1% SIG

                                      TABLE 2D    __________________________________________________________________________    HYBRID COMPARISON REPORT    VARIETY #1 = 38D66    VARIETY #2 = 3860    __________________________________________________________________________                   PRM BU  BU      TST SDG EST GDU               PRM SHD ACR ACR MST WT  VGR CNT SHD               ABS ABS ABS % MN                               % MN                                   ABS % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   91  95  154.2                           101 100 52.3                                       96  102 102           2   91  92  162.1                           106 100 52.0                                       108 95  99           LOCS               11  18  91  91  91  44  56  52  52           REPS               11  18  137 137 132 65  80  67  78           DIFF               0   3   7.9 5   1   0.2 13  7   3           PR > T               .999                   .000#                       .000#                           .000#                               .275                                   .155                                       .000#                                           .000#                                               .000#    __________________________________________________________________________               GDU STK PLT EAR RT  STA STK BRT DRP               SLK CNT HT  HT  LDG GRN LDG STK EAR               % MN                   % MN                       % MN                           % MN                               % MN                                   % MN                                       % MN                                           % MN                                               % MN    __________________________________________________________________________    TOTAL SUM           1   101 101 101 103 99  100 98  102 100           2   100 98  99  101 96  100 100 100 100           LOCS               21  120 43  43  37  50  78  22  21           REPS               31  190 69  69  52  69  114 31  30           DIFF               1   2   3   2   2   0   2   2   0           PR > T               .014+                   .000#                       .001#                           .315                               .299                                   .999                                       .077*                                           .004#                                               .999    __________________________________________________________________________               NLF GOS HD  GIB EYE ECB ECB               BLT WLT SMT ERS SPT DPE 1LF               ABS ABS ABS ABS ABS ABS ABS    __________________________________________________________________________    TOTAL SUM           1   7.5 8.3 100.0                           4.7 6.5 98.3                                       4.0           2   4.5 8.3 96.9                           4.0 6.3 96.2                                       4.0           LOCS               1   2   4   3   2   4   1           REPS               2   3   10  6   4   8   1           DIFF               3.0 0.0 3.1 0.7 0.3 2.1 0.0           PR > T  .999                       .182                           .423                               .500                                   .391    __________________________________________________________________________     * = 10% SIG     + = 5% SIG     # = 1% SIG

Strip Test Data for Hybrid 38D66

Comparison data was collected from strip tests that were grown byfarmers. Each hybrid was grown in strips of 4, 6, 8, 12, etc. rows infields depending on the size of the planter used. The data was collectedfrom strip tests that had the hybrids in the same area and weighed. Themoisture percentage was determined and bushels per acre was adjusted to15.5 percent moisture. The number of comparisons represent the number oflocations or replications for the two hybrids that were grown in thesame field in close proximity and compared.

Comparison strip testing was done between Pioneer Brand Hybrid 38D66 andPioneer Brand Hybrids 3860, 3861, 3893, and 38R21. The comparisons comefrom all the hybrid's adapted growing areas in the United States.

These results are presented in Table 3. As can be seen from the tablehybrid 38D66 has a yield advantage over all of the hybrids compared. Theaverage yield advantage is 3.5 bushels per acre. Hybrid 38D66demonstrated a significantly greater income advantage to the farmerbased on adjusted growth income over all hybrids compared. The averageincome advantage per acre was $5.44 with a high of $17.86. Hybrid38D66's yield and income advantage plus its advantage for othercharacteristics over these hybrids will make it an important additionfor most of the areas where these hybrids are grown.

                                      TABLE 3    __________________________________________________________________________    1997 PERFORMANCE COMPARISON REPORT FOR CORN    1 YEAR SUMMARY OF ALL STANDARD TEST TYPES                           Income/                               Pop Stand                                      Roots                                         Test    Brand      Product                   Yield                       Moist                           Acre                               K/Acre                                   (%)                                      (%)                                         Wt    __________________________________________________________________________    Pioneer    38D66                   142.6                       27.4                           319.38                               27.6                                   79 94 53.4    Pioneer    3860                   137.6                       26.8                           309.55                               26.9                                   78 96 52.9    Advantage      5.0 -.6 9.83                               .7  1  -2 .5    Number of Comparisons                   52  52  52  41  21 7  52    Percent Wins   60  42  56  49  48 0  65    Probability of Difference                   98  95  96  87  30 88 99    Pioneer    38D66                   132.8                       28.7                           294.77                               26.7                                   56 0  51.7    Pioneer    3861                   124.3                       28.7                           276.91                               25.0                                   88 0  50.7    Advantage      8.5 .0  17.86                               1.7 -32                                      0  1.0    Number of Comparisons                   3   3   3   3   1  0  3    Percent Wins   100 33  100 67  0  0  100    Probability of Difference                   85  0   97  80  0  0  99    Pioneer    38D66                   142.0                       24.5                           326.87                               27.4                                   85 86 53.6    Pioneer    3893                   137.5                       23.1                           320.45                               27.4                                   84 94 54.5    Advantage      4.5 -1.4                           6.42                               .0  1  -8 -.9    Number of Comparisons                   118 118 118 97  44 35 116    Percent Wins   65  23  54  46  45 9  17    Probability of Difference                   99  99  99  15  52 95 99    Pioneer    38D66                   141.8                       21.4                           334.66                               27.0                                   90 85 54.2    Pioneer    38R21                   140.9                       21.1                           334.09                               26.8                                   94 93 54.8    Advantage      .9  -.3 .57 .2  -4 -8 -.6    Number of Comparisons                   78  78  78  61  24 29 76    Percent Wins   51  40  49  49  29 28 20    Probability of Difference                   54  92  15  30  46 81 99    Pioneer    38D66                   141.9                       24.2                           327.35                               27.3                                   85 86 53.8    Weighted Avg   138.4                       23.3                           321.91                               27.1                                   85 94 54.2    Advantage      3.5 -.9 5.44                               .2  0  -8 -.4    Number of Comparisons                   251 251 251 202 90 71 247    Percent Wins   60  32  53  48  41 15 29    Probability of Difference                   99  99  99  77  63 98 99    __________________________________________________________________________     NOTE: The probability values are useful in analyzing if there is a "real"     difference in the genetic potential of the products involved. High values     are desirable, with 95% considered significant for real differences.

Characteristics of Pioneer Hybrid 38D66 are compared to Pioneer Hybrids3860, 38R21, 3893, 3861, and 37M81 in Table 4. The values given for mosttraits are on a 1-9 basis. In these cases 9 would be outstanding, while1 would be poor for the given characteristics. Table 4 shows that hybrid38D66 yields well for its maturity exhibits good drydown and goodstaygreen. Hybrid 38D66 also has better Northern Leaf Blight resistancethan any of the other hybrids. Hybrid 38D66's excellent yield combinedwith its other favorable agronomic characteristics should make it animportant hybrid to its area of adaptation.

Comparison of Key Characteristics for Hybrid 38D66

                                      TABLE 4    __________________________________________________________________________    Hybrid Patent Comparisons-Characteristics    Pioneer Hybrid 38D66 vs. Pioneer Hybrids 3860, 38R21, 3893, 3861,    __________________________________________________________________________    37M81             SILK                 PHY GDU GDU    VARIETY         CRM CRM CRM SILK                         PHY YLD H/POP                                     L/POP                                         D/D S/L R/L    __________________________________________________________________________    38D66         91  92  93  1150                         2210                             9   9   9   7   4   5    3860 92  92  92  1150                         2180                             9   8   9   6   4   4    38R21         92  91  90  1140                         2130                             9   9   8   5   6   3    3893 89  90  91  1130                         2160                             9   9   9   6   4   6    3861 93  96  95  1200                         2260                             7   7   7   6   5   3    37M81         97  97  98  1210                         2340                             9   9   8   7   4   4    __________________________________________________________________________         STA     TST     PLT EAR     BRT HSK GLF NLF    VARIETY         GRN D/T WT  E/G HT  HT  D/E STK CV  SPT BLT    __________________________________________________________________________    38D66         7   7   4   4   6   7   5   6   3       7    3860 7   7   4   6   7   7   6   6   5       3    38R21         4   8   5   8   6   8   6   6   7       5    3893 6   7   5   8   5   6   6   5   5       5    3861 3   7   4   4   8   7   6   7   6   5   4    37M81         6   7   4   6   6   7   5   4   6   4   5    __________________________________________________________________________         GOS ANT HD  GIB EYE ECB ECB    VARIETY         WLT ROT SMT ERS SPT 1LF 2SC    __________________________________________________________________________    38D66         8       8   4    3860 7       7   4   4   4   4    38R21         6       8   6       3   5    3893 7       8   5   4   3   4    3861 6       9   5   4   6   3    37M81         6       8   5   5   3   3    __________________________________________________________________________

Further Embodiments of the Invention

This invention includes hybrid maize seed of 38D66 and the hybrid maizeplant produced therefrom. The foregoing was set forth by way of exampleand is not intended to limit the scope of the invention.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants, orparts of plants, such as embryos, pollen, ovules, flowers, kernels,ears, cobs, leaves, seeds, husks, stalks, roots, root tips, anthers,silk and the like.

Duncan, Williams, Zehr, and widholm, Planta, (1985) 165: 322-332reflects that 97% of the plants cultured which produced callus werecapable of plant regeneration. Subsequent experiments with both inbredsand hybrids produced 91% regenerable callus which produced plants. In afurther study in 1988, Songstad, Duncan & Widholm in Plant Cell Reports(1988), 7: 262-265 reports several media additions which enhanceregenerability of callus of two inbred lines. Other published reportsalso indicated that "nontraditional" tissues are capable of producingsomatic embryogenesis and plant regeneration. K. P. Rao, et al., MaizeGenetics Cooperation Newsletter, 60: 64-65 (1986), refers to somaticembryogenesis from glume callus cultures and B. V. Conger, et al., PlantCell Reports, 6: 345-347 (1987) indicates somatic embryogenesis from thetissue cultures of maize leaf segments. Thus, it is clear from theliterature that the state of the art is such that these methods ofobtaining plants are, and were, "conventional" in the sense that theyare routinely used and have a very high rate of success.

Tissue culture of maize is described in European Patent Application,publication 160,390, incorporated herein by reference. Maize tissueculture procedures are also described in Green and Rhodes, "PlantRegeneration in Tissue Culture of Maize," Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 1982, at367-372) and in Duncan, et al., "The Production of Callus Capable ofPlant Regeneration from Immature Embryos of Numerous Zea MaysGeneotypes," 165 Planta 322-332 (1985). Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce maize plants having the genotype of 38D66.

Transformation of Maize

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modifed versions of native orendogenous genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreign,additional and/or modified genes are referred to herein collectively as"transgenes". Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transgenicversions of the claimed hybrid maize line 38D66.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used, alone or incombination with other plasmids, to provide transformed maize plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the maize plant(s).

Expression Vectors for Maize Transformation

Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e. inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art. One commonly used selectable marker gene forplant transformation is the neomycin phosphotransferase II (nptll) gene,isolated from transposon Tn5, which when placed under the control ofplant regulatory signals confers resistance to kanamycin. Fraley et al.,Proc. Natl. Acad. Sci. U.S.A., 80: 4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5: 299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3'-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet, 210: 86 (1987), Svab et al.,Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7: 171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) andStalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84: 131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol 1 15: 15la (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein "promoter" includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A "plantpromoter" is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as "tissue-preferred".Promoters which initiate transcription only in certain tissues arereferred to as "tissue-specific". A "cell type" specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An "inducible" promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of "non-constitutive" promoters. A"constitutive" promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inmaize. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in maize. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol.22: 361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90: 4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al.,Mol Gen. Genetics 243: 32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genet. 227: 229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inmaize or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in maize.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313: 810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol Biol 12: 619-632 (1989) andChristensen et al., Plant Mol. Biol. 18: 675-689 (1992)): pEMU (Last etal., Theor. Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBOJ. 3: 2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231: 276-285 (1992) and Atanassova et al., Plant Journal 2(3:291-300 (1992)).

The ALS promoter, a Xbal/Ncol fragment 5' to the Brassica napus ALS3structural gene (or a nucleotide sequence that has substantial sequencesimilarity to said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin maize. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in maize. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,-such as that from the phaseolin gene (Murai et al., Science 23: 476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11): 2723-2729(1985) and Timko et al., Nature 318: 579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genet. 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genet.224: 161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6: 217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5' and/or 3' region of a gene encoding the protein ofinterest. Targeting sequences at the 5' and/or 3' end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example, Becker etal., Plant Mol. Biol.20: 49 (1992), Close, P.S., Master's Thesis, IowaState University (1993), Knox, C., et al., "Structure and Organizationof Two Divergent Alpha-Amylase Genes From Barley", Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91: 124-129 (1989), Fontes etal., Plant Cell 3: 483-496 (1991), Matsuoka et al., Proc. Natl. Acad.Sci. 88: 834 (1991), Gould et al., J. Cell Biol 108: 1657 (1989),Creissen et al., Plant J. 2: 129 (1991), Kalderon, D., Robers, B.,Richardson, W., and Smith A., "A short amino acid sequence able tospecify nuclear location", Cell 39: 499-509 (1984), Stiefel, V.,Ruiz-Avila, L., Raz R., Valles M., Gomez J., Pages M.,Martinez-Izquierdo J., Ludevid M., Landale J., Nelson T., andPuigdomenech P., "Expression of a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation",Plant Cell 2: 785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is maize. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, and Simple Sequence Repeats (SSR) which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, METHODSIN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284 (CRC Press, BocaRaton,1993). Map information concerning chromosomal location is usefulfor proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes that Confer Resistance to Pests or Disease and that Encode

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(C) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(D) A vitamin-binding protein, such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by. Theapplication teaches the use of avidin and avidin homologues aslarvicides against insect pests.

(E) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm.163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No.5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(H) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(I) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(K) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(L) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

(M) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(P) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).

(R) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance To A Herbicide, For Example

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee etal.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al., Biol.Technology 10: 292 (1992) (production of transgenic plants that expressBacillus licheniformis α-amylase), Elliot et al., Plant Molec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes), S.oslashed.gaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Maize Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., "Procedures for Introducing Foreign DNA intoPlants" in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., "Vectors for PlantTransformation" in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant. Sci.10: 1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber et al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,591,616, issued Jan. 7,1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and maize. Hieiet al., The Plant Journal 6: 271-282 (1994); U.S. Pat. No. 5,591,616,issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., Trends Biotech. 6: 299(1988), Klein et al., Bio/Technology 6: 559-563 (1988), Sanford, J. C.,Physiol Plant 79: 206 (1990), Klein et al., Biotechnology 10: 268(1992). In maize, several target tissues can be bombarded withDNA-coated microprojectiles in order to produce transgenic plants,including, for example, callus (Type I or Type II), immature embryos,and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet199: 161 (1985) and Draper et al., Plant Cell Physiol.23: 451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51-61 (1994).

Following transformation of maize target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid maize plant.

Alternatively, a genetic trait which has been engineered into aparticular maize line using the foregoing transformation techniquescould be moved into another line using traditional backcrossingtechniques that are well known in the plant breeding arts. For example,a backcrossing approach could be used to move an engineered trait from apublic, non-elite line into an elite line, or from a hybrid maize plantcontaining a foreign gene in its genome into a line or lines which donot contain that gene. As used herein, "crossing" can refer to a simpleX by Y cross, or the process of backcrossing, depending on the context.

Industrial Applicability

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry.Stalks and husks are made into paper and wallboard and cobs are used forfuel and to make charcoal.

The seed of the hybrid maize plant and various parts of the hybrid maizeplant and transgenic versions of the foregoing, can be utilized forhuman food, livestock feed, and as a raw material in industry.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

Deposits

Applicant has made a deposit of at least 2500 seeds of Hybrid Maize Line38D66 with the American Type Culture Collection (ATCC), Manasss, Va.20110 USA. ATCC Deposit No. 203883. The seeds deposited with the ATCC onMar. 29. 1999 were taken from the deposit maintained by Pioneer Hi-BredInternational, Inc., 800 Capital Square, 400 Locust Street, Des Moines,Iowa 50309-2340 since prior to the filing date of this application. Thisdeposit of the Hybrid Maize Une 38D66 will be maintained in the ATCCdepository, which Is a public depository, for a period of 30 years, or 5years after the most recent request, or for the enforceable life of thePat., whichever Is longer, and will be replaced if it becomes nonviableduring that period. Additionally, Applicant has satisfied all therequirements of 37 C.F.R. §§1.801-1.809, including providing anIndication of the viability of the sample. Applicant Imposes norestrictions on the availability of the deposited material from theATCC; however, Applicant has no authority to waive any restrictionsimposed by law on the transfer of biological material or itstransportation in commerce. Applicant does not waive any infringement ofits rights granted under this patent.

What is claimed is:
 1. Hybrid maize seed designated 38D66,representative seed of said hybrid 38D66 having been deposited underATCC accession number
 203883. 2. A maize plant, or its parts, producedby the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovuleof the plant of claim
 2. 5. A tissue culture of regenerable cells of ahybrid maize plant 38D66, representative seed of said hybrid maize plant38D66 having been deposited under ATCC accession number 203883, whereinthe tissue regenerates plants capable of expressing all themorphological and physiological characteristics of said hybrid maizeplant 38D66.
 6. A tissue culture according to claim 5, the cells orprotoplasts being from a tissue selected from the group consisting ofleaves, pollen, embryos, roots, root tips, anthers, silks, flowers,kernels, ears, cobs, husks, and stalks.
 7. A maize plant, or its parts,regenerated from the tissue culture of claim 5 and capable of expressingall the morphological and physiological characteristics of hybrid maizeplant 38D66, representative seed having been deposited under ATCCaccession number
 203883. 8. A hybrid maize plant according to claim 2,wherein the genetic material of said plant contains one or moretransgenes.
 9. A maize plant, or its parts, wherein at least oneancestor of said maize plant is the maize plant, or its parts, of claim2, said maize plant expressing a combination of traits selected from thegroup consisting of: high yield for grain, high yield for silage, veryhard grain texture, good drydown behavior, good late season plant healthand is well suited to the Northcentral United States, Canada, Maritimeand Central Europe.
 10. A method for developing a maize plant in a maizeplant breeding program using plant breeding techniques which includeemploying a maize plant, or its parts, as a source of plant breedingmaterial, comprising: using the maize plant, or its parts, of claim 2 asa source of said breeding material.
 11. The maize plant breeding programof claim 10 wherein plant breeding techniques are selected from thegroup consisting of: recurrent selection, backcrossing, pedigreebreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, and transformation.
 12. A method fordeveloping a maize plant in a maize plant breeding program using plantbreeding techniques, which include employing a maize plant, or itsparts, as a source of plant breeding material, comprising: using themaize plant, or its parts, of claim 8 as a source of said breedingmaterial.
 13. The maize plant breeding program of claim 12 wherein plantbreeding techniques are selected from the group consisting of: recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.